A “Proteomic Ruler” for Protein Copy Number and Concentration Estimation without Spike-in Standards

Absolute protein quantification using mass spectrometry (MS)-based proteomics delivers protein concentrations or copy numbers per cell. Existing methodologies typically require a combination of isotope-labeled spike-in references, cell counting, and protein concentration measurements. Here we present a novel method that delivers similar quantitative results directly from deep eukaryotic proteome datasets without any additional experimental steps. We show that the MS signal of histones can be used as a “proteomic ruler” because it is proportional to the amount of DNA in the sample, which in turn depends on the number of cells. As a result, our proteomic ruler approach adds an absolute scale to the MS readout and allows estimation of the copy numbers of individual proteins per cell. We compare our protein quantifications with values derived via the use of stable isotope labeling by amino acids in cell culture and protein epitope signature tags in a method that combines spike-in protein fragment standards with precise isotope label quantification. The proteomic ruler approach yields quantitative readouts that are in remarkably good agreement with results from the precision method. We attribute this surprising result to the fact that the proteomic ruler approach omits error-prone steps such as cell counting or protein concentration measurements. The proteomic ruler approach is readily applicable to any deep eukaryotic proteome dataset—even in retrospective analysis—and we demonstrate its usefulness with a series of mouse organ proteomes.Mass spectrometry (MS)¹ is now capable of analyzing the proteome to considerable depth, and more than 10,000 proteins have been reported in single mammalian cell types (1). In the past decade, MS-based proteomics has gone from sole identification to the quantification of proteins, which has typically meant relative quantification between samples (2–4). Apart from the presence of a protein and its relative fold changes between different conditions (5), it is often desirable to estimate absolute quantities such as molar concentrations or copy numbers per cell, which can be compared for different proteins (6). For instance, in systems biology, even a rough estimate of the copy number can help to establish initial parameters for simulation (7). Likewise, clinical protein measurements are typically done in absolute terms of titers, such as milligrams per deciliter. For this purpose various approaches have been utilized, including correlating total MS signals to visualized structures in the cell (8) and extrapolating from spiked-in reference protein mixtures (9) or from endogenous proteins quantified via accurately characterized, isotopically labeled peptide (10) or protein fragment standards (11). Absolute quantification is then achieved through quantification relative to a known reference. In all cases, results scale with the amount of input material or amount of spiked-in standard. Accurate protein concentration measurements are thus an essential and often limiting factor for overall accuracy. Commonly used dye-based protein determination methods rely on the reactivity of few amino acid residues—mainly tryptophan and tyrosine (12) in the case of the Lowry and BCA assays, or a hydrophilic/hydrophobic balance of the proteins in the case of Bradford reagent (13). Systematic errors of up to a factor of 2 may therefore arise from the selection of a non-optimal protein standard (14). An additional, often ignored source of errors is the cross-reactivity of the reagents with non-proteinaceous cell components such as thiols, nucleic acids, and phospholipids.To convert protein quantities to copies per cell, all methods require knowledge of the number of cells used for the analysis. This can be obtained directly via cell counting or indirectly through knowledge of the total protein amount per cell, which in turn is a function of cell volume and total protein concentration. However, cells are not necessarily uniform; therefore scaling by cell numbers may be inaccurate, as a 25% variation of the diameter of a sphere-shaped cell corresponds to a 2-fold change in cell volume. In tissues, not only are cell sizes variable, but visual counting of cells is also problematic. For instance, up to 5-fold differences in calculated cell volumes have been reported for enterocytes of the intestinal mucosa (15).Any deviations in protein determination or cell counts will inevitably carry over to the final readout, even when very precise MS methods are used. This limits the overall accuracy, without showing up as a decrease in the precision of the quantification, as measured by standard deviations or coefficients of variation.In the course of studying the colon cancer proteome, we recently devised a method for estimating absolute amounts of individual proteins or protein classes based on the proportion of their MS signals to the total MS signal (16). We termed the method the Total Protein Approach, because we relate this proportion to a total protein mass. To obtain copy numbers, we specifically used the total protein mass per cell, which needs to be determined or estimated separately.In this study, we expanded the method by a concept we call the “proteomic ruler” to further allow correct absolute scaling of the readout without additional steps. We made use of the defined amount of genetic information in each cell, encoded in a known amount of DNA. We show that an accurate determination of the DNA content in a proteomic sample helps to directly determine the number of cells. We then demonstrate that the MS signal derived from histones, around which DNA is wrapped in a defined ratio, can be used as a natural standard in a whole proteome dataset. It serves as a proteomic ruler that allows the estimation of total protein amounts per cell. Thereby the quantitative readout can be absolutely scaled to copies per cell without the need for cell counting or protein concentration determination.     

China Council for International Cooperation on Environment and Development(CCICED)

1ImportanceofChina'sbiodiversityDefinition:BiodiversityistakentoincludetheentiregeneticcontentandallecologicalfunctionsprocessesandstructuresofthefaunaandfloraofChina.Overview:China'sbiodiversityisthethirdrichestintheworld,andricherthananyotherAsianortemperatecountry.Thiswealthofspeciesandpopulationsisexplainedbyverylargesize,extremeclimaticandgeographicvariation,longcontinentalstabilityandthemostextensivesubtropicalzoneintheworld.Majorecosystemsinclude1)forests,thatrangefromtemperatetotropi…     

What about cultural ecosystems? Opportunities for cultural considerations in the “International Standards for the Practice of Ecological Restoration”

The Society for Ecological Restoration's 2016 (SER) “International Standards for the Practice of Ecological Restoration” is a living document intended to guide restoration projects “anywhere in the world.” Given its intended global scope and in hopes of informing future editions, this document is critically assessed in light of the role people have played in ecosystems around the world. We argue that the Standards has an underlying nature–culture dichotomization that limits its applicability; in qualifying what it calls “cultural ecosystems” for rehabilitation, rather than restoration, the Standards privileges colonial visions of ecological restoration. We also discuss the Standards' representation of the ecological impacts and practices of indigenous groups. Whereas the Standards claims that preindustrial cultural ecosystems exist in states similar to unmodified areas, many historians, anthropologists, and paleoecologists would point out that preindustrial people sometimes had massive environmental impacts through agriculture, hydrological engineering, over‐hunting, living in dense urban environments, transporting species, burning on a scale capable of changing the climate, and other practices. Furthermore, the Standards does not discuss how the cultural goals of indigenous groups fit into the overall picture of ecological restoration. Future drafts of the Standards should more accurately frame the diverse roles people play in nature, and create global standards that account for the validity of cultural goals for ecological restoration.     

History and evolution of the International Association for Plant Biotechnology (IAPB): 1963–2008

The International Association for Plant Biotechnology (IAPB) was founded in 1963 at the first truly international conference on plant tissue culture, which was organized by Philip R. White. White was a devoted internationalist and was strongly committed to global scientific cooperation. He felt that the time had come for the international tissue culture community to organize so that it could meet regularly and provide a forum to its members for the exchange of ideas and information of mutual interest and use. The various activities of the IAPB since its founding—the publication of its newsletter, its journal, and the proceedings of its quadrennial congresses—faithfully document the remarkable advances in plant biotechnology that were made possible by the successful integration of tissue culture and molecular biology. In particular, the congress proceedings serve as time capsules, providing a wealth of information about the best of science and the most prominent scientists of the time. The history of the IAPB is indeed the history of plant biotechnology.     

IMGT®, the International ImMunoGeneTics Information System® for Immunoinformatics

IMGT, the International ImMunoGeneTics information system ( http://imgt.cines.fr ), was created in 1989 by the Laboratoire d'ImmunoGénétique Moléculaire (LIGM) (Université Montpellier 2 and CNRS) at Montpellier, France, in order to standardize and manage the complexity of immunogenetics data. IMGT is recognized as the international reference in immunogenetics and immunoinformatics. IMGT is a high quality integrated knowledge resource, specialized in (i) the immunoglobulins (IG), T cell receptors (TR), major histocompatibility complex (MHC) of human and other vertebrates; (ii) proteins that belong to the immunoglobulin superfamily (IgSF) and to the MHC superfamily (MhcSF); and (iii) related proteins of the immune systems (RPI) of any species. IMGT provides a common access to standardized data from genome, proteome, genetics, and three-dimensional (3D) structures for the IG, TR, MHC, IgSF, MhcSF, and RPI. IMGT interactive on-line tools are provided for genome, sequence, and 3D structure analysis. IMGT Web resources comprise 10,000 HTML pages of synthesis and knowledge (IMGT Scientific chart, IMGT Repertoire, IMGT Education, etc.) and external links (IMGT Bloc-notes and IMGT other accesses).     

International conference on “Photosynthesis Research for Sustainability-2016”

During June 19–26, 2016, an international conference (http://photosynthesis2016.cellreg.org/) on “Photosynthesis Research for Sustainability-2016” was held in honor of Nathan Nelson and Turhan Nejat Veziro?lu at the Institute of Basic Biological Problems, Russian Academy of Sciences, formerly Institute of Photosynthesis, Academy of Sciences of the USSR, Pushchino, Russia. Further, this conference celebrated the 50th anniversary of the Institute. We provide here a brief introduction and key contributions of the two honored scientists, and then information on the conference, on the speakers, and the program. A special feature of this conference was the awards given to several young investigators, who are recognized in this Report. Several photographs are included to show the excellent ambience at this conference. We invite the readers to the next conference on “Photosynthesis and Hydrogen Energy Research for Sustainability-2017”, which will honor A.S. Raghavendra (of University of Hyderabad), William Cramer (of Purdue University) and Govindjee (of University of Illinois at Urbana-Champaign); it will be held during the Fall of 2017 (from October 30 to November 4), at the University of Hyderabad, Hyderabad, India. See <https://prs.science>.     

International Law and Western Sahara’s Maritime Area

Abstract

Western Sahara’s coastal waters have become contentious because of seabed petroleum exploration and fisheries undertaken pursuant to treaties between Morocco and the European Union, Japan, and Russia. These activities have been protested by the territory’s government-in-exile, the Saharawi Arab Democratic Republic. In 2017 Morocco announced its intention to adopt legislation to create an exclusive economic zone (EEZ) on the territory’s coast. This article considers the status of Saharan coastal waters in the circumstances of decolonization and occupation. The obligations on states interested in exploring and extracting Saharan ocean resources are considered and are argued to be restrictive regardless of the status of the territory’s coastal waters and recognition of a Saharawi state.     

Editorial Committee of Vertebrata PalAsiatica

主　编Editor in Chief　张弥曼ZHANGMi Man (CHANGMee mann)副主编DeputyEditorinChief　李锦玲LIJin Ling编　委MembersofEditorialCommitteePhilipJ.CURRIE (RoyalTyrrellMuseumofPalaeontology,Drumheller,Canada)邓　涛DENGTao (InstituteofVertebratePaleontologyandPaleoanthropology,CAS)PhilippeJANVIER (L .A .1 2duC .N .R .S .,InstitutdePal啨ｏｎｔｏｌｏｇｉｅ,Paris,France)李传夔LIChuan Kui (InstituteofVertebratePaleontologyandPaleoanthropology,CAS)李锦玲LIJin Ling (Instituteo…     

Building International Indigenous People’s Partnerships for Community-Driven Health Initiatives

In this article we present an international Indigenous people’s partnership project co-led by two Indigenous communities, Musqueam (Coast Salish, Canada) and Totoras (Quichua, Ecuador), as a community-driven health initiative. The Musqueam-Totoras partnership includes Indigenous organizations, universities, international agencies, government, and nongovernmental organizations to address Indigenous health concerns in both communities. Our collaborative approach provides a framework to (a) increase the development expertise of Indigenous people internationally, (b) increase skills among all participants, and (c) facilitate Indigenous knowledge mobilization and translation to promote cultural continuity. This international Indigenous people’s partnership between north and south reflects the diversity and commonalities of Indigenous knowledge, contributes to cultural revitalization, and minimizes the impact of assimilation, technology, and globalization. Indigenous people’s partnerships contribute to self-determination, which is a prerequisite to the building and maintenance of healthy communities and the promotion of social justice. The exchange of Indigenous knowledge upholds Indigenous values of respect, reciprocity, relevance, and responsibility. Given the history of colonization and the negligence of governments in the exercising of these values with respect to Indigenous communities, this contemporary exchange among Indigenous people in the Americas serves to reclaim these values and practices. International cooperation empowering Indigenous people and other marginalized groups has become fundamental for their advancement and participation in globalized economies. An international Indigenous people’s partnership provides opportunities for sharing cultural, historical, social, environmental, and economic factors impacting Indigenous health. These partnerships also create beneficial learning experiences in community-based participatory research and community-driven health initiatives, provide culturally sensitive research ethics frameworks, increase capacity building, and address basic human needs identified by participating communities.     

Do Short International Layovers Allow Sufficient Opportunity for Pilots to Recover?

For Australian pilots, short layovers (<40 h) are a feature of many international patterns. However, anecdotal reports suggest that flight crew members find patterns with short slips more fatiguing than those with a longer international layover, as they restrict the opportunity to obtain sufficient sleep. The current study aimed to determine whether pilots operating international patterns with short layovers have sufficient opportunity to recover prior to the inbound flight. Nineteen international pilots (ten captains, nine first officers) operating a direct return pattern from Australia to Los Angeles (LAX) with a short (n=9) 9±0.8 h (mean±S.D) or long (n=10) 62.2±0.9 h LAX layover wore an activity monitor and kept a sleep/duty diary during the pattern. Immediately before and after each flight, pilots completed a 5 min PalmPilot‐based psychomotor vigilance task (Palm‐PVT). Flights were of comparable duration outbound (3.5±0.6 h) and inbound (14.3±0.6 h) and timing. The amount of sleep obtained in‐flight did not significantly vary as a function of layover length. However, pilots obtained significantly more sleep during the inbound (3.7±0.8 h) than the outbound flight (2.2±0.8 h). Pilots with the shorter layover obtained significantly less sleep in total during layover (14.0±2.7 h vs. 19.6±2.5), due to significantly fewer sleep periods (3.0±0.7 vs. 4.0±0.9). However, neither mean sleep duration nor the sleep obtained in the 24 h prior to the inbound flight significantly differed as a function of layover length. Response speed significantly varied across the pattern, and a significant interaction was also observed. For pilots with a short layover, response speed was significantly slower at the end of both the outbound and inbound flight, and prior to the inbound flight (i.e., at the end of layover), relative to response speed at the start of the pattern (pre‐trip). Similarly, response speed for the longer layover was slower at the end of the outbound flight compared to pre‐trip (approaching significance, p=0.073). However, response speed at the beginning of the inbound flight was significantly faster than pre‐trip and did not significantly differ from pre‐trip at the end of the inbound flight. The data suggest that short slips (<40 h) do not allow pilots the opportunity to obtain sufficient sleep to reverse the effects of fatigue accumulated during the outbound flight. As a result, their response speed prior to the inbound flight is substantially slower than the response speed of flight crew with a longer layover.